GFP

fig 1. A still image from gfpdimer.pdb. (GFP,
though a monomer in vivo, crystalizes as a dimer.) Click to view
(requires Rasmol).

Green Fluorescent Protein
as a Reporter Gene

By Ben Buxton

"If you don't already use GFP, you will. Maybe not today,
maybe not tomorrow, but soon... and for the rest of your life"

Green-fluorescent protein (GFP), one of the hottest new biological
tools, is responsible for the stunning bioluminescence of the Pacific Northwest
Jellyfish, Aequorea victoria. While in its native host, GFP emits a green colored
light when it accepts blue light from a calcium activated photoprotein. Given
another source of blue light, such as UV, which cannot be detected by human
eyse, GFP will produce a green light. A human using a UV light will therefore
only see GFP (and other fluorescents), because it will be the only source of
visible light. It is just this property that makes the clone of the GFP gene
a powerful tool for use in the lab. Since the cloning of the GFP gene in 1992
by Douglas Prasher (of USDA/APHIS) it has been demonstrated that the GFP gene
can be expressed in non-homologous species, from yeast and plant cells to Drosophila
and vertebrates, including humans1. It seems that this lack of specificity
to jellyfish is because GFP requires no substrates (other than oxygen) to fluoresce,
and has no physiological effect on cell operation. In a sense a transformed
cell doesnÕt ÒknowÓ that GFP is even there, and therefore
can remain alive while being studied.

The main concern of this paper, however, is with the application
of GFP as a reporter gene. Reporter genes are common biological tools that
demonstrate clearly when they a gene is turned on or off. For instance,
the Lac-Z gene (coding for Beta-Galactosidase), when uninterrupted and
functional, will change the colorless substrate X-gal to blue. If functional
Lac-Z is part of a plasmid in a bacteria or yeast colony, it will change
the cells from from white to blue. This so called blue white selection
is commonly used to find cells with recombinant DNA or plasmids (cells
with an insert interrupting Lac-Z), after performing a transformation (introducing
new DNA into a cell). GFPÕs distinctive properties allow it also
to be used as a reporter of gene expression.

Natures method of gene expression, where the proteins
that make up a cell are made from their DNA codes, gives molecular biologists
a useful tool. Each gene in an organism is located after the promoter and
inducer sequences specific to it. These are portions of DNA occurring before
the actual coding sequences that an RNA polymerase enzyme binds to in order
to begin translation of the gene. In a little understood process, certain
promoters have a property which makes them bind more often to RNA polymerase
then other promoters, and thus proteins are expressed in different amounts.
In other words, the cell does not really ÒknowÓ what genes
it is translating, it only ÒknowsÓ which promoters are being
used. Since the cell only sees the promoter, the gene for GFP can be attached
downstream from the promoter (and inducer) DNA sequences for whatever gene
one wishes to study. If this DNA construct is introduced into a cell, whenever
the gene under study is expressed in the cell, the GFP produced simultaneously
will fluoresce green under a UV lamp. Rather than having to detect the
protein of interest, say with antibodies, or by various other destructive
methods you can watch the living, normally functioning cell and clearly
determine when the gene is turned on and off.

The following series of figures illustrate how GFP could
be used to "report" when a gene encoding a fetal form of globin,
a hemoglogin subunit, is being expressed: (figure by Ben Buxton)

Using GFP as a reporter gene is not without its potential
problems. A major one is that only one gene can be tracked at a time. To
solve this problem, reserachers are mutating the chromophore, or light
emitting portion of the protein, in attempts to find a mutant form of the
gene that would fluoresce in a different color. In addition, GFP signals
can be weak and hard to detect without high expression. Some cells will
naturally fluoresce, too, such as lignin in plant cells. This could make
bona fide GFP fluorescence hard to detect.